Combined assessment of transgenic rodent gene mutation and micronuclei formation by benzo[a]pyrene, N-ethyl-N-nitrosourea, 2,4-diaminotoluene and ethyl carbamate.

This study combined an in vivo Transgenic Rodent Gene Mutation Assay (TGRA) and Mammalian Erythrocyte Micronucleus Test (MNT) using male MutaMouse to assess the four positive control substances recommended by the OECD test guideline no. 488: benzo[a]pyrene (B[a]P), N-ethyl-N-nitrosourea (ENU), 2,4-diaminotoluene (2,4-DAT), and ethyl carbamate (EC). The studies followed the procedures described in the OECD guidelines 488 and 474. Gene mutations were assessed in site-of-contact organs, glandular stomach and small intestine, as well as in the liver, and germ cells isolated from testes and cauda epididymis. Micronuclei were measured in peripheral blood reticulocytes by flow cytometry. B[a]P and ENU induced dose-dependent increases in gene mutations in somatic and germ cells, as well as significant increases in the micronucleus rate in peripheral blood reticulocytes. In contrast, 2,4-DAT and EC induced weak gene mutation responses in some somatic tissues, but no significant effects in germ cells. This highlights the tissue-specific responses and thus a need for differentiated assessments. All test substances induced an increase of micronuclei to different extents in peripheral blood reticulocytes. The combined TGRA-MNT protocol enabled comprehensive evaluation of gene mutation and micronucleus induction within the same study, supporting reduction in animal use for in vivo mutagenicity testing. B[a]P and ENU can be used as positive control substances for the combined TGRA-MNT approach.